The primary objectives of this research are to determine, by application of multidisciplinary approaches, the molecular basis for the specificity of action of potential anticancer agents, those lipophilic diaminopyrimidines and pteridines, that act as inhibitors of the enzyme dihydrofolate reductase (DHFR), and to understand the explicit mechanisms of their antineoplastic activity. These goals will be achieved through the (1) determination of the X-ray crystal structures of selected members from four antifolate classes (lipophilic diaminopyrimidines, conformationally flexible 5-benzyl diaminopyrimidines, soluble diaminotriazines, and side-chain modified methotrexate analogues), (2) description of their molecular and electronic properties from empirical molecular mechanics and ab initio molecular orbital energy minimization techniques, and (3) development of models that describe the specific binding interactions at the active site of the inhibitor-enzyme complex. This study will provide data that can identify the stable conformational isomers of DHFR inhibitors, delineate their differing degrees of flexibility, determine the influence of specific substituents upon conformation, and define the nature of the active site interactions with these inhibitors. It is well documented that the general requirement for tight binding of inhibitors to DHFR from any species is a 2,4-diaminopyrimidine or s-triazine ring. The binding selectivity and specificity of inhibitors for DHFR enzymes from different species are demonstrated from data showing significant changes in inhibitory action with antifolates having only a single substituent change. Thus, the introduction of a 6-methyl in 5-benzyl diaminopyrimidine greatly enhances its activity against plasmodial enzymes, but diminishes it against bacterial enzymes. Similarly, within a given series of lipophylic 5-adamantyl diaminopyrimidines, a single change in the 6-position results in a 500-fold enhancement of its potency to inhibit mammalian DHFR. These compounds are currently in clinical trial as potential anticancer agents. Although methotrexate is the most widely used chemotherapeutic agent, its limitations include poor cellular uptake, failure to cross the blood-brain barrier and frequent occurrence of cell resistance. In contrast, lipophilic antifolates readily diffuse through cell membranes and cross the blood-brain barrier. However, their insolubility in physiological media and their central nervous system toxicity are major problems in their use. Therefore, a detailed knowledge of these antifolates at the molecular level is required to understand their activities.